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de novo sequencing is the term used to describe the initial sequence analysis performed to obtain the primary genetic sequence of a particular organism. A detailed genetic analysis of an organism is possible only after de novo sequencing has been performed.

For de novo sequencing using capillary electrophoresis, the target DNA is fragmented and cloned into a viral or plasmid vector. Cloning provides amplification of the target DNA (by bacterial growth) and allows sequencing primers to bind to known sequence in the vector and extend the sequence into the unknown target DNA. de novo sequencing methods are described in the Sequencing Chemistry Guide page 16-20.

Step-by-Step Guide to de novo Sequencing

DNA Purification kit

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

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PCR Kit

Amplify the DNA region of interest.

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DNA strands

Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products. The products are then injected into a capillary. All current Applied Biosystems DNA sequencing kits use cycle sequencing protocols.

Select the BigDye® chemistry right for the sequencing application.

Sequencing specificity

BigDye®
Direct

BigDye®
Terminator V3.1

BigDye®
Terminator V1.1

All types of sequencing xx xx xx
High resolution close to the sequencing primer xx xx
(if using POP-6™)
M13 tailed PCR primers and M13 sequencing primer xx † x x
Universal tail primer other than M13 primer x x
Long read (>650 bp) x xx x
Mixed base (heterozygote) with ratio 10/50 to 50/50 xx x xx
GC-rich, GT-rich, difficult template* x x x

x   Satisfactory
xx  Optimal
–   Not Recommended

BigDye® Direct, BigDye® Terminator V3.1, and BigDye® Terminator V1.1 will provide good sequence quality for the majority of these templates. In some cases, the dGTP BigDye® terminators have been specially designed for these difficult templates and will provide better results

†  BigDye® Direct requires the use of M13 tailed primer for the PCR reaction. This kit contains BigDye® Direct PCR Master Mix, BigDye® Direct Sequencing Master Mix to perform the PCR cleanup and sequencing reaction in a one-step reaction, BigDye® Direct M13 Fwd Primer (5′ TGTAAAACGACGGCCAGT 3′), BigDye® Direct M13 Rev Primer (5′ CAGGAAACAGCTATGACC 3′), and Control DNA CEPH 1347-02.

* Why use M13 tailed PCR primer or other universal tail primer?
Use of M13 or universal primers in a sequencing workflow streamlines the workflow, and reduces the possibility of human error.

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Sequencing Reaction Cleanup

Before capillary electrophoresis, sequencing reaction needs to be purify to remove the fluorescent ddNTP.

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Which Sample Cleanup Product Is Right for You?

Cleanup Method Description & Application Product
Reagent-Based Sequencing Template CleanupScavenges all unincorporated BigDye® terminators. Stabilizes samples before analysis. Best for long and short fragment recovery.
Capillary Electrophoresis

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

Which Electrophoresis Instrument (Genetic Analyzer) Is Right for You?

  Instrument
 3730xl
3730xL
3730
3730
3500xl
3500xL
3500
3500
3130xl
3130xL
3130
3130
310
310
Number of Capillaries96482481641
Compatible Applications:
(S) Supported; (A) AB Demonstrated; (C) Customer Demonstrated; (N) Not Supported
De Novo Sequencing SSSSSSS

After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required to translate the raw data into the corresponding electropherogram.

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Highlighted products for de novo Sequencing: